Solid-phase extraction of small biologically active peptides on cartridges and microelution 96-well plates from human urine.

Currently liquid chromatography - mass spectrometry (LC-MS) analysis after solid-phase extraction (SPE) on weak cation-exchange cartridges is a method of choice for anti-doping analysis of small bioactive peptides such as growth hormone releasing peptides (GHRPs), desmoporessin, LHRH, and TB-500 short fragment. Dilution of urine samples with phosphate buffer for pH adjustment and SPE on weak cation exchange microelution plates was tested as a means to increase throughput of this analysis. Dilution using 200 mM phosphate buffer provides good buffering capacity without affecting the peptides recoveries. SPE on microelution plates was performed on Waters Positive Pressure-96 Processor with subsequent evaporation of eluates in nitrogen flow. Though the use of smaller sample volume decreases the pre-concentration factor and increases the limits of detection of 5 out of 17 detected peptides, the recovery, linearity, and reproducibility of the microelution extraction were comparable with cartridge SPE. The effectiveness of protocols was confirmed by analysis of urine samples containing ipamorelin, and GHRP-6 and its metabolites. SPE after urine sample dilution with buffer can be used for faster sample preparation. The use of microelution plates decreases consumption of solvents and allows processing of up to 96 samples simultaneously. Cartridge SPE with manual рН adjustment remains the best option for confirmation. Copyright © 2015 John Wiley & Sons, Ltd.

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides.

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.

Preparation, purification, and characterization of a reversibly lipidized desmopressin with potentiated anti-diuretic activity.

PURPOSE: . To prepare and characterize a reversibly lipidized dipalmitoyl desmopressin (DPP), and to compare its anti-diuretic efficacy and biodistribution with that of unmodified desmopressin (DDAVP). METHODS: Dithiothreitol (DTT) was used to reduce the intramolecular disulfide bond in DDAVP, and the reduced DDAVP was treated with a thiopyridine-containing disulfide lipidization reagent, Pal-CPD. The product, DPP, was purified by acid precipitation and, subsequently, by size-exclusion chromatography. Reversed-phase HPLC was used to analyze the purity and to evaluate the hydrophobicity of the product. Mass spectrometry was employed to characterize its molecular structure. The biological activity of DPP was demonstrated by the antidiuretic effects in vasopressin-deficient Brattleboro rats. Preliminary pharmacokinetic and biodistribution studies of intravenously injected DDAVP and DPP were carried out in CF-1 mice. RESULTS: DDAVP was readily reduced by a 2-fold molar excess of DTT at 37 degrees C for 0.5 hr. DPP was formed by the reaction of reduced DDAVP with Pal-CPD. Each DPP molecule contains two palmitic acid moieties, which link to the peptide via two disulfide bonds. After acid precipitation and size-exclusion chromatography, the purity was found to be approximately 95%, and the overall yield was 57%. When DPP was administered subcutaneously to Brattleboro rats, the potency of the anti-diuretic activity of DDAVP was enhanced to more than 250-fold. The plasma concentration of intravenously injected DDAVP in mice decreased rapidly during the first 20 min and followed by a slow elimination rate. However, in DPP administered mice, the plasma concentration actually increased in the first 20 min, followed by a slow elimination with a rate similar to that in DDAVP-injected mice. The regeneration of DDAVP was detected in the plasma of mice treated with DPP. Studies of the organ distribution in mice indicated that the liver retention of DPP was longer than that of DDAVP. On the other hand, the intestinal excretion of DPP was significantly less than that of DDAVP. CONCLUSIONS: The 250-fold increase of the anti-diuretic potency in DPP is most likely due to a slow elimination and prolonged tissue retention, together with the regeneration of active DDAVP, in the animals. Our results indicate that reversible lipidization is a simple and effective approach for improving the efficacy of many peptide drugs.

[Characterization of FVIII/VW circulating after DDAVP administration (author's transl)]. / Caracterización del FVIII/VW circulante tras la administración de DDAVP.

These studies were designed with the purpose of providing more information relevant about the new FVIII/VW circulating after administration intranasally DDAVP. One health subject was administered 260 microliter total dose of DDAVP. The newly released FVIII/VW could not be distinguished form circulating FVIII/VW on the basis of molecular size, electrophoretic mobility or "in vitro" stability of VIII:C and VIIIR:AG despite apparent differences in the duration of response of the procoagulant and antigenic components "in vivo". FVIII/VW cryoethanol-precipitations studies showed similar increase in the high molecular weight (HMW) and low molecular weight (LMW) after DDAVP administration. This suggest a immediately polymerization in the new VIIIR:AG circulating after DDAVP administration.